The occurrence and genomic characteristics of the blaIMI-1 carbapenemase-producing Enterobacter cloacae complex retrieved from natural water sources in central Thailand.

AIM: Carbapenem resistance among Enterobacteriaceae is a serious threat to humans worldwide. This study aims to evaluate the phenotypic and genotypic characterization of carbapenemase-producing Enterobacter cloacae complex (ECC) retrieved from water sources in the central part of Thailand. METHODS AND RESULTS: Samples were collected from water bodies surrounding farms and communities in central Thailand. The species were identified by using MALDI-TOF MS. The minimum inhibitory concentration (MIC) and antibiotic susceptibility were determined. The carbapenemase-producing genes were detected by PCR and whole genome sequencing (WGS). ECC with chromosome-encoded blaIMI-1 carbapenemase were detected. These isolates were resistant to last-resort antibiotics such as carbapenems and colistin as well as penicillin. In addition, all blaIMI-1 genes isolated from this study were found to be associated with chromosomally integrated Xer-dependent integrative mobile elements (IMEXs). CONCLUSION: These findings highlight the diversity and dissemination of carbapenemases-producing Enterobacterales in environmental sources. With the increasing detection of carbapenemase genes worldwide, we should be aware of the blaIMI-producing E. cloacae complex with a high resistance profile and the ability to mobilize within the environment.

Allergen components of Dermatophagoides farinae recognised by serum immunoglobulin (Ig)E in Thai dogs with atopic dermatitis.

BACKGROUND: Dermatophagoides farinae (Der f) is a common allergen in dogs with atopic dermatitis (AD). However, the relevant components of Der f require further investigation. OBJECTIVES: We aimed to provide data on the immunoglobulin (Ig)E-binding specific components of Der f for further diagnostic and therapeutic applications. ANIMALS: Serum samples were collected from five healthy, nine Der f-allergic atopic and seven non-Der f-allergic atopic dogs identified based on an intradermal skin test. METHODS AND MATERIALS: We explored the component profiles of Der f extracts through 2D gel electrophoresis and IgE immunoblotting. The IgE-binding components in both groups of atopic dogs were analysed by mass spectrometry. RESULTS: The majority of Der f-allergic atopic dogs recognised Der f Alternaria alternata allergen 10 (Der f Alt a 10), elongation factor 1-alpha (EF1-α), gelsolin-like allergen Der f 16, Der f 28 and Der f 2. Der f 3, Der f 10, Der f 20 and Der f 32 were recognised as minor allergens. Alpha-enolase, serine protease, arginine kinase and a few hypothetical proteins were recognised components in both groups of atopic dogs. Unexpectedly, Der f 15 (chitinase) was found to be a minor component. CONCLUSIONS AND CLINICAL IMPORTANCE: Multiple IgE-binding allergens of Der f were identified in Thai atopic dogs. We propose that the specific antigen set that is bound by IgE, comprising Der f Alt a 10, EF1-α, gelsolin-like Der f 16, Der f 28 and Der f 2, could be used for future diagnostics and immunotherapy platforms.

Leptospira infection and shedding in dogs in Thailand.

BACKGROUND: Leptospirosis is a widespread zoonosis and has been recognized as a re-emerging infectious disease in humans and dogs, but prevalence of Leptospira shedding in dogs in Thailand is unknown. The aim of this study was to determine urinary shedding of Leptospira in dogs in Thailand, to evaluate antibody prevalence by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA), and to assess risk factors for Leptospira infection. In Northern, Northeastern, and Central Thailand, 273 stray (n = 119) or client-owned (n = 154) dogs from rural (n = 139) or urban (n = 134) areas were randomly included. Dogs that had received antibiotics within 4 weeks prior to sampling were excluded. No dog had received vaccination against Leptospira. Urine was evaluated by real-time polymerase chain reaction (PCR) specific for lipL32 gene of pathogenic Leptospira. Additionally, urine was cultured for 6 months in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. Antibodies were measured by ELISA and MAT against 24 serovars belonging to 15 serogroups and 1 undesignated serogroup. Risk factor analysis was performed with backwards stepwise selection based on Wald. RESULTS: Twelve of 273 (4.4%; 95% confidence interval (CI): 2.0-6.8%) urine samples were PCR-positive. In 1/273 dogs (0.4%; 95% CI: 0.01-1.1%) Leptospira could be cultured from urine. MAT detected antibodies in 33/273 dogs (12.1%; 95% CI: 8.2-16.0%) against 19 different serovars (Anhoa, Australis, Ballum, Bataviae, Bratislava, Broomi, Canicola, Copenhageni, Coxi, Grippotyphosa, Haemolytica, Icterohaemorrhagiae, Khorat, Paidjan, Patoc, Pyrogenes, Rachmati, Saxkoebing, Sejroe). In 111/252 dogs (44.0%; 95% CI: 37.9-50.2%) immunoglobulin M (IgM) and/or immunoglobulin G (IgG) antibodies were found by ELISA. Female dogs had a significantly higher risk for Leptospira infection (p = 0.023). CONCLUSIONS: Leptospira shedding occurs in randomly sampled dogs in Thailand, with infection rates comparable to those of Europe and the USA. Therefore, the potential zoonotic risk should not be underestimated and use of Leptospira vaccines are recommended.

Antibody levels to Malassezia pachydermatis and Staphylococcus pseudintermedius in atopic dogs and their relationship with lesion scores.

BACKGROUND: Elevated immunoglobulin E (IgE) levels to Malassezia or Staphylococcus species in human atopic dermatitis are related to the skin severity index; a similar association has not been reported in atopic dogs. OBJECTIVES: To investigate serum levels of allergen-specific IgE, total specific IgG and IgG subclasses (IgG1 and IgG2) for M. pachydermatis and S. pseudintermedius, and to correlate them with the severity of dermatitis in dogs. ANIMALS: Serum samples were collected from dogs categorized by age and disease status. Groups 1 and 2: <3-year-old healthy (n = 9) and atopic dogs (n = 9), respectively; and groups 3 and 4: ≥3-year-old healthy (n = 11) and atopic dogs (n = 14), respectively. METHODS AND MATERIALS: Antibody levels were measured by ELISA. The Canine Atopic Dermatitis Lesion Index (CADLI) was analyzed in relation to antibody levels. RESULTS: Specific IgE and total IgG against M. pachydermatis and S. pseudintermedius were significantly increased in atopic dogs of all ages. Although differences between atopic and healthy dogs, with regard to specific IgG1 and IgG2 levels to each microbe, varied in significance within age groups. No significant relationships were found between the CADLI and any specific immunoglobulin levels for both microbe types. CONCLUSIONS AND CLINICAL IMPORTANCE: In dog skin, microbes may act as allergens triggering inflammatory responses via IgE- and IgG-dependent pathway(s). The affinity of the IgG subclass produced may vary according to antigen type. Specific IgE levels may be related to clinical disease in dogs and not to skin lesion severity.

Correction to: Genomic analysis of Leptospira interrogans serovar Paidjan and Dadas isolates from carrier dogs and comparative genomic analysis to detect genes under positive selection.

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Genomic analysis of Leptospira interrogans serovar Paidjan and Dadas isolates from carrier dogs and comparative genomic analysis to detect genes under positive selection.

BACKGROUND: Leptospirosis is an emerging infectious disease worldwide that can cause high morbidity and mortality rates in humans and animals. The causative spirochetes have reservoirs in mammalian hosts, but there has been limited analysis of the genomes of isolates recovered from animals. The aims of this study were to characterize genomic features of two Leptospira interrogans strains recently isolated from asymptomatic dogs in Thailand (strains CUDO5 and CDUO8), and to perform comparative genome analyses with other strains. Molecular adaptive evolution in L. interrogans as signaled by positive selection also was analyzed. RESULTS: Whole genome sequence analysis revealed that strains CUDO5 and CUDO8 had genome sizes of approximately 4.9 Mbp with 35.1% GC contents. Using monoclonal antibodies, strains CUDO5 and CUDO8 were identified as serovars Paidjan and Dadas, respectively. These strains harbored genes known to be associated with acute and chronic infections. Using Single Nucleotide Polymorphisms phylogeny (SNPs) with 97 L. interrogans strains, CUDO5 and CUDO8 had closest genetic relatedness with each other. Nevertheless, the serovar determinant region (rfb locus) showed variations in the genes encoding sugar biosynthesis. Amongst 13 representative L. interrogans strains examined for molecular adaptive evolution through positive selection under the site-model of Phylogenetic Analysis of Maximum Likelihood, genes responsible for iron acquisition (tlyA and hbpA), motility (fliN2, flgK, and flhB) and thermal adaptation (lpxD1) were under increased selective pressure. CONCLUSIONS: L. interrogans serovar Paidjan strain CUDO5 and serovar Dadas strain CUDO8 had close genetic relatedness as analyzed by SNPs phylogeny. They contained genes with established roles in acute and chronic leptospirosis. The rfb locus in both serovars showed gene variation associated with sugar biosynthesis. Positive selection analysis indicated that genes encoding factors involved in motility, temperature adaptation, and iron acquisition were under strong positive selection in L. interrogans. These may be associated with adaptation in the early stages of infection.

Autochthonous lactic acid bacteria isolated from pig faeces in Thailand show probiotic properties and antibacterial activity against enteric pathogenic bacteria.

Lactic acid bacteria (LAB) play an important role in pig health and performance that arises from their beneficial impacts on the balance of gastrointestinal microbes, ability to fight enteric pathogens, and capacity to support the immune system. The aim of this study was to evaluate the functional and safety aspects of five previously isolated autochthonous LAB strains, (Lactobacillus plantarum 22F, 25F and 31F, Pediococcus acidilactici 72N and Pediococcus pentosaceus 77F) from pig faeces as potential probiotics for a pig feed supplement. The functional and safety properties of the strains were assessed by in vitro tests. The functional properties tested were their abilities in tolerating low pH values under simulated gastric conditions, their cell surface properties (hydrophobicity, auto- and co-aggregation), antibacterial activity against the common enteric pathogenic bacteria in pigs (such as pathogenic Escherichia coli, Salmonella Choleraesuis and Streptococcus suis), and diacetyl production. The safety of the strains was analyzed based on the absent of haemolysis on blood and bile salt hydrolase activity. Although all strains demonstrated diacetyl production, good survivability and antibacterial activities, L. plantarum 22F and 25F showed the best performance with the strongest antibacterial actions against the indicator pathogens. Of the strains, only P. pentosaceus 77F exhibited haemolysis or bile salt hydrolase activity. Furthermore, a principal component analysis revealed that L. plantarum 22F possessed superior functional and safety aspects compared to the other four autochthonous strains and to reference strains L. plantarum JCM 1149 and P. acidilactici DSM 20284. Further in vivo studies using oral administration of the strains are justified to assess their effectiveness as feed supplements for pigs.

Nasal carriage of methicillin-resistant Staphylococcus pseudintermedius in dogs treated with cephalexin monohydrate.

This study aimed to investigate the nasal carriage of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs treated with oral cephalexin monohydrate. Ten dogs with superficial pyoderma were monitored longitudinally for carriage of MRSP for up to 1 year after treatment; the strains were typed and antibiograms were determined. Methicillin-susceptible S. pseudintermedius (MSSP) was recovered prior to treatment in all dogs and could be isolated after 12 months in 1 dog. Methicillin-resistant Staphylococcus pseudintermedius was detected within 1 week of treatment in all dogs, and 3 clones represented by ST45, ST112, and ST181 were consistently present for up to 12 months after treatment. All MRSP isolates were resistant to at least 7 common antimicrobials. Oral cephalexin monohydrate treatment selected for strains of multi-resistant MRSP, which were still present after 1 year.

Portage nasal deStaphylococcus pseudintermediusrésistant à la méthicilline chez les chiens traités à l'aide de céphalexine monohydrate. Cette étude visait à étudier le portage nasal de Staphylococcus pseudintermedius résistant à la méthicilline (SPRM) chez les chiens traités à l'aide de céphalexine monohydrate par voie orale. Dix chiens ayant une pyodermie superficielle ont été surveillés dans une étude longitudinale pour le portage de SPRM pendant jusqu'à un an après le traitement; les souches ont été typées et des antibiogrammes ont été réalisés. Staphylococcus pseudintermedius susceptible à la méthicilline (SPSM) a été récupéré avant le traitement chez tous les chiens et pouvait être isolé jusqu'à 12 mois chez un chien. Staphylococcus pseudintermedius résistant à la méthicilline a été détecté une semaine après le traitement chez tous les chiens et 3 clones représentés par ST45, ST112 et ST181 étaient continuellement présents jusqu'à 12 mois après le traitement. Tous les isolats de SPRM étaient résistants à au moins sept antimicrobiens communs. Le traitement à la céphalexine monohydrate par voie orale a été choisi pour les souches multirésistantes de SPRM qui étaient toujours présentes après un an.(Traduit par Isabelle Vallières).

Characterization of a Novel Composite Staphylococcal Cassette Chromosome mec in Methicillin-Resistant Staphylococcus pseudintermedius from Thailand.

A novel staphylococcal cassette chromosome mec (SCCmec) composite island (SCCmecAI16-SCCczrAI16-CI) was identified in Staphylococcus pseudintermedius. Four integration site sequences for SCC subdivided the 60,734-bp island into 41,232-bp SCCmecAI16, 19,400-bp SCCczrAI16, and 102-bp SCC-likeAI16 elements. SCCmecAI16 represents a new combination of ccrA1B3 genes with a class A mec complex. SCCczrAI16 contains ccrA1B6 and genes related to restriction modification and heavy metal resistance. SCCmecAI16-SCCczrAI16-CI was found in methicillin-resistant S. pseudintermedius sequence type 112 (ST112) and ST111 isolated from dogs and veterinarians in Thailand.

Water-soluble microencapsulation using gum Arabic and skim milk enhances viability and efficacy of Pediococcus acidilactici probiotic strains for application in broiler chickens.

OBJECTIVE: This study aimed to develop and evaluate the effectiveness of a water-soluble microencapsulation method for probiotic strains using gum Arabic (GA) and skim milk (SKM) over a three-month storage period following processing. METHODS: Four strains of Pediococcus acidilactici (BYF26, BYF20, BF9, and BF14) that were typical lactic acid bacteria (LAB) isolated from the chicken gut were mixed with different ratios of GA and SKM as coating agents before spray drying at an inlet temperature 140°C. After processing, the survivability and probiotic qualities of the strains were assessed from two weeks to three months of storage at varied temperatures, and de-encapsulation was performed to confirm the soluble properties. Finally, the antibacterial activity of the probiotics was assessed under simulated gastrointestinal conditions. RESULTS: As shown by scanning electron microscopy, spray-drying produced a spherical, white-yellow powder. The encapsulation efficacy (percent) was greatest for a coating containing a combination of 30% gum Arabic: 30% skim milk (w/v) (GA:SKM30) compared to lower concentrations of the two ingredients (p<0.05). Coating with GA:SKM30 (w/v) significantly enhanced (p<0.05) BYF26 survival under simulated gastrointestinal conditions (pH 2.5 to 3) and maintained higher survival rates compared to non-encapsulated cells under an artificial intestinal juices condition of pH 6. De-encapsulation tests indicated that the encapsulated powder dissolved in water while keeping viable cell counts within the effective range of 106 for 6 hours. In addition, following three months storage at 4°C, microencapsulation of BYF26 in GA:SKM30 maintained both the number of viable cells (p<0.05) and the preparation's antibacterial efficacy against pathogenic bacteria, specifically strains of Salmonella. CONCLUSION: Our prototype water-soluble probiotic microencapsulation GA:SKM30 effectively maintains LAB characteristics and survival rates, demonstrating its potential for use in preserving probiotic strains that can be used in chickens and potentially in other livestock.

Genomic characterization of carbapenem and colistin-resistant Klebsiella pneumoniae isolates from humans and dogs.

Introduction: Carbapenem and colistin-resistant Enterobacteriaceae, including Klebsiella pneumoniae, have become a growing global concern, posing a significant threat to public health. Currently, there is limited information about the genetic background of carbapenem and colistin-resistant K. pneumoniae isolates infecting humans and dogs in Thailand. This study aimed to characterize carbapenem and colistin-resistant genes in six resistant K. pneumoniae clinical isolates (three from humans and three from dogs) which differed in their pulse field gel electrophoresis profiles. Methods: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), antimicrobial susceptibility testing, and whole-genome sequencing were employed to identify and analyze the isolates. Results and discussion: All six isolates were carbapenemase-producing K. pneumoniae isolates with chromosomally carried blaSHV, fosA, oqxA and oqxB genes, as well as nine to 21 virulence genes. The isolates belonged to five multilocus sequence types (STs): one isolate from a human and one from a dog belonged to ST16, with the other two human isolates being from ST340 and ST1269 and the other two dog isolates were ST147 and ST15. One human isolate and two dog isolates harbored the same blaOXA-232 gene on the ColKP3 plasmid, and one dog isolate carried the blaOXA-48 gene on the IncFII plasmid. Notably, one human isolate exhibited resistance to colistin mediated by the mcr-3.5 gene carried on the IncFII plasmid, which co-existed with resistance determinants to other antibiotics, including aminoglycosides and quinolones. In conclusion, this study provides a comprehensive characterization of both chromosome- and plasmid-mediated carbapenem and colistin resistance in a set of K. pneumoniae clinical isolates from unrelated humans and dogs in Thailand. The similarities and differences found contribute to our understanding of the potential widescale dissemination of these important resistance genes among clinical isolates from humans and animals, which in turn may contribute to outbreaks of emerging resistant clones in hospital settings.

Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs.

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.

Novel pseudo-staphylococcal cassette chromosome mec element (ψSCCmec57395) in methicillin-resistant Staphylococcus pseudintermedius CC45.

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I-pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to ß-lactams (mecA; blaZ), aminoglycosides [aac(6')-Ie-aph(2')-Ia; aph(3')-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (ΨSCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ΨSCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ΨSCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ΨSCCmec57395 element amplified by long-range PCR revealed the presence of ΨSCCmec57395 in the 33 additional isolates of MRSP CC45. The ΨSCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand.

Antifungal agent susceptibilities and interpretation of Malassezia pachydermatis and Candida parapsilosis isolated from dogs with and without seborrheic dermatitis skin.

Malassezia pachydermatis and Candida parapsilosis are recognized as commensal yeasts on the skin of healthy dogs but also causative agents of eborrheic dermatitis, especially in atopic dogs. We determined and compared the susceptibility levels of yeasts isolated from dogs with and without seborrheic dermatitis (SD) using the disk diffusion method (DD) for itraconazole (ITZ), ketoconazole (KTZ), nystatin (NYS), terbinafine (TERB) and 5-fluorocytosine (5-FC) and the broth microdilution method (BMD) for ITZ and KTZ. The reliability between the methods was assessed using an agreement analysis and linear regression. Forty-five M. pachydermatis and 28 C. parapsilosis isolates were identified based on physiological characteristics and an approved molecular analysis. By DD, all tested M. pachydermatis isolates were susceptible to ITZ, KTZ, NYS and TERB but resistant to 5-FC. Only 46 - 60% of the tested C. parapsilosis isolates were susceptible to KTZ, TERB and 5-FC, but ITZ and NYS were effective against all. By BMD, over 95% of M. pachydermatis isolates were susceptible to KTZ and ITZ with an MIC90 < 0.03 and 0.12 µg/ml, respectively. The frequency of KTZ- and ITZ-resistant C. parapsilosis was 29% and 7%, and the MIC90 values were 1 µg/ml and 0.5-1 µg/ml, respectively. Regarding the agreement analysis, 2.2% of minor errors were observed in M. pachydermatis and 0.2-1% of very major errors occurred among C. parapsilosis. There were no significant differences in the yeast resistance rates between dogs with and without SD. KTZ and ITZ were still efficacious for M. pachydermatis but a high rate of KTZ resistant was reported in C. parapsilosis.

Comparative genomic analysis of Colistin resistant Escherichia coli isolated from pigs, a human and wastewater on colistin withdrawn pig farm.

In this study, genomic and plasmid characteristics of Escherichia coli were determined with the aim of deducing how mcr genes may have spread on a colistin withdrawn pig farm. Whole genome hybrid sequencing was applied to six mcr-positive E. coli (MCRPE) strains isolated from pigs, a farmworker and wastewater collected between 2017 and 2019. Among these, mcr-1.1 genes were identified on IncI2 plasmids from a pig and wastewater, and on IncX4 from the human isolate, whereas mcr-3 genes were found on plasmids IncFII and IncHI2 in two porcine strains. The MCRPE isolates exhibited genotypic and phenotypic multidrug resistance (MDR) traits as well as heavy metal and antiseptic resistance genes. The mcr-1.1-IncI2 and IncX4 plasmids carried only colistin resistance genes. Whereas, the mcr-3.5-IncHI2 plasmid presented MDR region, with several mobile genetic elements. Despite the MCRPE strains belonged to different E. coli lineages, mcr-carrying plasmids with high similarities were found in isolates from pigs and wastewater recovered in different years. This study highlighted that several factors, including the resistomic profile of the host bacteria, co-selection via adjunct antibiotic resistance genes, antiseptics, and/or disinfectants, and plasmid-host fitness adaptation may encourage the maintenance of plasmids carrying mcr genes in E. coli.

Bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage with or without antibiotics in a tropical environment.

BACKGROUND: In tropical environments, boar semen is prepared either from a boar on the same farm as the sow herd or collected in semen collection centers and then transported to other farms. Thus, the semen doses can be used for artificial insemination either immediately or preserved for 2-3 days. The present study investigated the bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage in semen extender with or without antibiotics in Thailand. M&M: In total, 20 Duroc ejaculates were collected. Each ejaculate was diluted in Beltsville Thawing Solution extender either with 0.25 g of gentamicin per liter (ANTIBIOTIC) or without gentamicin (NO-ANITIBIOTIC) to create semen doses containing 3,000 × 106 sperm/100 mL. These were stored at 17 °C for 4 days. Semen characteristics and total bacterial count (CFU per mL, log10) were measured after collection and during storage. RESULTS: Sperm viability was decreased by 6.4% for every 1.0 log10 increase in total bacterial count (p = 0.026) and Staphylococcus spp. were the most frequently isolated across ejaculates. Throughout the 4 days of storage, sperm motility, viability and acrosome integrity in the ANTIBIOTIC group were higher than those in the NO-ANTIBIOTIC group (p < 0.05), while the total bacterial count was lower (1.9 ± 0.1 versus 3.9 ± 0.1 log10, respectively; p < 0.001). Without antibiotic supplementation, the total numbers of bacteria counted on days 2 and 3 of storage were higher than those determined on days 0 and 1 (p < 0.001). Differences in semen quality were detected on days 2 and 3 between the NO-ANTIBIOTIC and ANTIBIOTIC groups in high-viability semen (p < 0.05). However, no differences in sperm quality between the NO-ANTIBIOTIC and ANTIBIOTIC groups were detected in the low-viability semen on each storage day (p > 0.05). On the last day of preservation, Globicatella sanguinis (57.2%), Delftia acidovorans (18.9%) and Micrococcus spp. (5.9%) remained as the top three most abundant contaminants in the semen with antibiotic. CONCLUSION: Our findings contribute new insights toward reducing antibiotics as well as rational antibiotic use in the boar AI industry. The growth of bacteria was significantly greater only after 2 days of preservation in the semen without antibiotic. For semen doses diluted from highly viable ejaculates, it is possible to store for 2 days without any antibiotic supplementation. Moreover, bacterial counts increased at the end of storage in the presence of gentamycin, suggesting the loss of bacteriostatic properties of gentamicin to the growth of bacteria during storage.

Potential natural antimicrobial and antibiofilm properties of Piper betle L. against Staphylococcus pseudintermedius and methicillin-resistant strains.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper betle L. has potent of antimicrobial activity and is widely used as a traditional remedy to treat skin infections. However, no clear evidence exists concerning antimicrobial and antibiofilm activity against Staphylococcus pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) opportunistic pathogens that cause wound infections and pyoderma in canines and zoonotic disease. AIM OF THE STUDY: The antimicrobial and antibiofilm activities of P. betle extract were assessed against S. pseudintermedius and MRSP strains. MATERIALS AND METHODS: Ethanol leaf extract of P. betle was investigated for its antibacterial effect on S. pseudintermedius and MRSP by broth microdilution and time-kill assays. Biofilm inhibition and production assays were performed to evaluate antibiofilm and biofilm eradication effects, respectively. Biofilm-associated gene expression was further studied using real-time polymerase chain reaction (PCR). The possible interaction between IcaA and major compounds in P. betle was analyzed by molecular docking. RESULTS: The extract showed minimum inhibitory concentration (MIC) at 250 µg/mL. Growth inhibition of P. betle at 1 MIC against the bacteria was initially observed after treatment for 4 h. All isolates were completely killed after 18 h exposure to the extract. Minimum biofilm inhibitory concentrations (MBICs) of the extract against the tested isolates ranged 1/2 MIC to 1 MIC, while minimum biofilm eradication concentration (MBEC) of P. betle was initialed at 8 MIC. Quantitative inhibition and eradication effects were observed in representative strains. The extract at 1/2 MIC and 1 MIC values inhibited biofilm formation up to 100%, with bacterial biofilm removed at up to 94.21% by 4 MIC of the extract. The extract downregulated the expression of the icaA gene among biofilm-producing isolates. The most abundant compounds, 4-allyl-1,2-diacetoxybenzene and eugenol showed a strong affinity with IcaA protein at -5.65 and -5.31 kcal/mol, respectively. CONCLUSIONS: P. betle extract demonstrated the antibacterial, antibiofilm, and biofilm-removal activity against S. pseudintermedius and MRSP. Downregulation of the icaA gene expression and protein interaction were possible modes of action of the extract that impacted biofilm production. This extract showed promise as an alternative treatment for S. pseudintermedius infection, especially drug-resistant and biofilm-associated cases.

Boar Seminal Microbiota in Relation to Sperm Quality under Tropical Environments.

The present study was carried out to determine the seminal microbiota of boars and their correlation with sperm quality. A total of 17 ejaculates were collected from 17 Duroc boars and were classified according to sperm quality into two groups: low-quality (n = 8) and high-quality (n = 9). Each ejaculate was subjected to (i) semen evaluation, (ii) bacterial culture and MALDI-TOF identification, and (iii) 16S rRNA gene sequencing and bioinformatic analyses. No difference in the total bacterial count, alpha diversity, and beta diversity between the high-quality group and the low-quality group was detected (p > 0.05). While Globicatella sanguinis was negatively correlated with sperm quality (p < 0.05), Delftia acidovorans was positively correlated with sperm quality (p < 0.05). Lactobacillales (25.2%; LB) and Enterobacterales (10.3%; EB) were the most dominant bacteria and negatively correlated: EB = 507.3 - 0.5 × LB, R2 = 0.24, p < 0.001. Moreover, the abundance of Escherichia-shigella was negatively correlated with LB (r = -0.754, p < 0.001) and positively correlated with Proteus (r = 0.533, p < 0.05). Alysiella was positively correlated with Lactobacillus (r = 0.485, p < 0.05), Prevotella (r = 0.622, p < 0.01), and Staphylococcus (r = 0.489, p < 0.05). In conclusion, seminal microbiota is significantly associated with boar semen qualities. The distributions of the most dominant bacterial genera, the differences in the abundance of small subset microbes, and their correlation appear to have far more impact than the overall seminal bacterial content (e.g., total bacterial count, alpha diversity, and beta diversity) on sperm quality.

Rapid identification of canine uropathogens by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and the clinical factors that correlated bacterial species and antimicrobial resistance.

Using the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) method for bacterial diagnosis, rapid urine sample preparation can reduce time relapsing of diagnosis and improve discriminatory power in coinfection cases. We aimed to evaluate rapid urine preparation procedures before MALDI-TOF MS application using dog clinical urine samples in comparison with standard microbiological diagnostic methods by agreement analysis. We determined the frequency and distribution of bacteria and bacterial resistance and their correlations to clinical history. Three experimental procedures comprising direct centrifugation, 10% sodium dodecyl sulfate digestion, and ultrasonic preparation were performed for method validation and sensitivity. Sterile urine containing Escherichia coli and/or Staphylococcus aureus were used as simulated samples. By ultrasonic preparation, the microorganisms could be detected 1.46-1.51 × 105 CFU, which was considered the most suitable technique. This preparation was significantly consistent with the routine method based on data from Hospital Information Systems for 50 urine samples from canine cystitis. By standard protocol, Enterobacteriaceae and Staphylococcus pseudintermedius were found in most of the 155 urine samples with cystitis. Extended-spectrum beta-lactamase-producing Enterobacteriaceae was found in 25-30% of the samples. Imipenem resistance was found in 70% of Acinetobacter baumannii cases; almost all were resistant to second-generation fluoroquinolones and tetracyclines. The most efficient antibiotic for treating bacterial urinary tract infection was amoxicillin plus clavulanic acid. A. baumannii and Pseudomonas aeruginosa were susceptible to pradofloxacin. Prolonged urine catheterization was linked to lower urinary tract infections by Enterobacter spp., which also correlated with chronic kidney disease.

Selection and evaluation of lactic acid bacteria from chicken feces in Thailand as potential probiotics.

Background: Lactic acid bacteria (LAB) are widely used as probiotics in poultry production due to their resilience to low pH and high bile salt concentrations, as well as their beneficial effects on growth performance and antagonistic activity against enteric pathogens. However, the efficacy of probiotics depends on strain selection and their ability to colonize the host's intestine. This study aimed to select, identify, and evaluate LAB strains isolated from chicken feces in Thailand for potential use as probiotics in the chicken industry. Methods: LAB strains were isolated from 58 pooled fresh fecal samples collected from chicken farms in various regions of Thailand, including commercial and backyard farms. Gram-positive rods or cocci with catalase-negative characteristics from colonies showing a clear zone on MRS agar supplemented with 0.5% CaCO3 were identified using MALDI-TOF mass spectrometry. The LAB isolates were evaluated for acid (pH 2.5 and pH 4.5) and bile salt (0.3% and 0.7%) tolerance. Additionally, their cell surface properties, resistance to phenol, antimicrobial activity, hemolytic activity, and presence of antimicrobial resistance genes were determined. Results: A total of 91 LAB isolates belonging to the Pediococcus, Ligilactobacillus, Limosilactobacillus, and Lactobacillus genera were obtained from chicken feces samples. Backyard farm feces exhibited a greater LAB diversity compared to commercial chickens. Five strains, including Ligilactobacillus salivarius BF12 and Pediococcus acidilactici BF9, BF14, BYF20, and BYF26, were selected based on their high tolerance to acid, bile salts, and phenol. L. salivarius BF12 and P. acidilactici BF14 demonstrated strong adhesion ability. The five LAB isolates exhibited significant cell-cell interactions (auto-aggregation) and co-aggregation with Salmonella. All five LAB isolates showed varying degrees of antimicrobial activity against Salmonella strains, with P. acidilactici BYF20 displaying the highest activity. None of the LAB isolates exhibited beta-hemolytic activity. Whole genome analysis showed that L. salivarius BF12 contained ermC, tetL, and tetM, whereas P. acidilactici strains BF9 and BF14 carried ermB, lnuA, and tetM. Conclusion: The selected LAB isolates exhibited basic probiotic characteristics, although some limitations were observed in terms of adhesion ability and the presence of antibiotic resistance genes, requiring further investigation into their genetic location. Future studies will focus on developing a probiotic prototype encapsulation for application in the chicken industry, followed by in vivo evaluations of probiotic efficacy.